SBIR Phase I: Novel Microarray Platforms For Detection Of Rare Molecules In Complex Mixtures

这个小企业创新研究（SBIR）一期项目提出构建一个具有高蛋白质结合能力的新型微阵列平台，以增强荧光检测。现有微阵列表面的局限性包括基于平台的光学干扰和有限或无效的生物分子结合能力。这些限制了现有微阵列表面上结合反应的最终灵敏度。我们将以两种方式构造复合曲面或修饰曲面。首先，将硝化纤维素（一种能够不可逆地结合许多不同生物分子的聚合物）浇铸在光学透明的多孔蚀刻膜上。我们相信这种新表面将保持两种起始材料的一些特性。通过改变轨迹蚀刻膜的孔隙结构，我们将优化所得到的膜捕获复杂蛋白质混合物的能力，并允许敏感的荧光检测。其次，我们将直接用功能性硅烷修饰轨迹蚀刻膜，以提供允许蛋白质和核酸共价偶联的化学基团。这种方法将被证明有利于保持原有轨迹蚀刻结构的光学兼容性。这种类型的修饰，光学透明的轨迹蚀刻膜可能是抗体阵列的最佳选择，其中捕获的分子可以以足够的密度固定以提供敏感的分析表面。该项目的更广泛的影响/商业潜力将是为一种新的分析工具提供基础，该工具将允许建立基于抗体和抗原的灵敏度更高的分析方法。它还将提供一种创新的表面，以定量捕获复杂混合物的生化成分，从而允许检测稀有分子。微阵列在生物科学研究、疾病和药物发现过程以及人类和动物诊断中发挥着越来越重要的作用。它们提供了从少量珍贵的临床和研究样本中提取多个值所需的并行处理工具。具有增强灵敏度的微阵列将允许检测可能参与细胞调节，细胞分化和疾病机制的稀有生物分子。逆相蛋白阵列（RPPA）对于了解多种疾病状态下的细胞变化非常重要。例如，在癌症中，可以在表面上发现少量肿瘤细胞的裂解物，然后用许多不同的抗体进行询问，以阐明这些肿瘤细胞群中的蛋白质表达模式。通过拥有一个能够支持最敏感分析的平台，这些技术的能力将得到显著增强。

This Small Business Innovation Research (SBIR) Phase I project proposes to construct a new microarray platform with high protein binding capacity that allows for enhanced fluorescence detection. Limitations of existing microarray surfaces include platform-based optical interferences and limited or ineffective binding capacity for biomolecules. These limit the ultimate sensitivity of binding reactions mounted on existing microarray surfaces. We will construct a composite or modified surface in two ways. First, by casting nitrocellulose (a polymer able to bind many different biomolecules essentially irreversibly) on an optically transparent porous track-etched membrane. We believe this new surface will maintain some properties of both starting materials. By varying the pore structure of the track-etched membrane, we will optimize the resulting membranes ability to capture complex protein mixtures and permit sensitive fluorescent detection. Secondly, we will directly modify the track-etched membrane with functional silanes to provide chemical groups permitting covalent coupling of proteins and nucleic acids. This approach should prove beneficial to maintain the optical compatibility of the original track-etched structure. This type of modified, optically transparent track-etched membrane may be optimal for antibody arrays where the capture molecule can be immobilized at a sufficient density to provide a sensitive assay surface. The broader impact/commercial potential of this project will be to provide the basis for a new analytical tool that will allow establishment of antibody- and antigen-based assays of enhanced sensitivity. It also will provide an innovative surface to capture quantitatively the biochemical components of complex mixtures in such a way as to permit the detection of rare molecules. Microarrays play an increasingly important role in bioscience research, disease, and drug discovery processes as well as in human and animal diagnostics. They provide parallel processing tools required to extract multiple values from small amounts of precious clinical and research samples. Microarrays with enhanced sensitivity will permit the detection of rare biomolecules that may be involved in cellular regulation, cellular differentiation, and disease mechanisms. Reverse phase protein arrays (RPPA) are important for understanding cellular changes in a variety of disease states. In cancer, for example, lysates from small numbers of tumor cells can be spotted on a surface and then interrogated with many different antibodies to elucidate protein expression patterns in these tumor cell populations. The power of these techniques will be enhanced significantly by having a platform able to support the most sensitive assays.